The HslVU protease-chaperone complex is a two component, proteasome-related system typical of eubacteria and well-known in eukaryotes. It is involved in intracellular degradation of a number of important proteins including transcription factors, cell cycle regulatory proteins and short-lived proteins. The HslVU is composed of multiple copies of two heat shock proteins, the 19 kDa HslV peptidase and the 50 kDa HslU, the ATPase and chaperone. The HslV is an N-terminal threonine protease related to the β-subunits of the 20S proteasome from eukaryotes and the archaebacteria. The HslV forms a barrel-shaped dodecameric complex by stacking two hexameric rings of HslV subunits and each of the HslV subunit contains an N-terminal Thr active site for proteolysis (FIG. 1). In the HslV dodecamer, the intra-ring interfaces are mainly stabilized by polar interactions whereas conserved hydrophobic residues are engaged to form the inter-ring interface. As a member of AAA-ATPase superfamily, the HslU demonstrates ATPase and unfoldase/chaperone activities simultaneously and related to the base part of the proteasomal 19S complex.
In the HslVU complex, the central pores of HslU and HslV are aligned, so that HslU transfers substrate poypeptides through the pores into inner proteolytic chamber of HslV. The HslV alone shows a very weak peptidase activity towards carbobenzoxy-Gly-Gly-Leu-7-amido-4-methyl coumarin (Z-GGL-AMC), a small fluorogenic peptide substrate, but its activity increases 1-2 orders of magnitude when it binds to HslU in the presence of ATP12. The HslU has increased affinity for HslV in the presence of protein substrate.
In the HslvU complex, the Hs1U carboxy-terminal octapeptide EDLSRFIL (SEQ ID NO 3) (termed as HslU C-tail) is intercalated into a cleft between adjacent Hslv subunits with a network of interactions. Several polar amino acid residues (Lys28 and Arg35) and hydrophobic residues (Phe54) of Hslv form electrostatic and hydrophobic interactions respectively with side chain and main chain atoms of C-tail residues (FIG. 1). This C-tail insertion is accompanied by a conformational change in the active site of Hslv which resulted in allosteric activation of proteolytic activity of HslV. Therefore, the HslU C-tail acts as allosteric activator of Hslv protease. The observation that Hslv can be activated by synthetic peptides comprising the HslU carboxy-terminal sequence (C-tail) confirmed the role of this peptide in allosteric activation as well as in HslvU complex formation.
Orthologues of prokaryotic HslV and HslU in parasitic protozoa i.e. Trypnosoma brucei (causative agent of sleeping sickness), Plasmodium falciparum (causative agent of malaria) and Leishmania species (causative agent of leishmaniasis) are novel drug target candidates. The genes homologous to protozoal HslVU are not present in human genome. Therefore, intracellular protein degradation by activation of HslVU system has been considered as a innovative strategy for development of new antiparasitic agents. No other molecule is known to be capable of activating HslV in the absence of its natural activator, HslU. We have identified quanazoline and chromone derivatives as HslV activators in the presence of HslU.